Physiological and anatomical studies of development of superficial axodendritic synaptic pathways in neocortex

EXPERIMENTAL

NEUROLOGY

2, 324-347 (1960)

Physiological and Anatomical Studies of Development of Superficial Axodendritic Synaptic Pathways in Neocortex D.P. Departments

PURPURA, of Neurological

Surgeons,

M. W. Surgery

Columbia

CARMICHAEL,

AND

E. M.

and Pharmucology, University, New York,

Received

March

4,

HOLJSEPIAN~

College of Physicians New York

and

1960

Neocortical axodendritic synaptic organizations involved in the production and spread of evoked superficial cortical responses (SCR’s) have been studied in kittens ranging in age from the late fetal stage to 6 weeks. Electrophysiological data are correlated with the salient morphological features of cortical neurons at various developmental stages. Graded, long-duration (SO-80 msec) SCR’s are detectable 5 to 7 mm from the site of stimulation during the first postnatal week and 8 to 10mm after this period. Short-duration (IO-20msec) responses, similar to those recorded in mature animals, are detectable in perinatal animals at the site of stimulation but not at distances greater than 2 mm from stimulating electrodes until the third postnatal week. Linear changes in latency with distance are observed at all stages of development. Distant SCR’s exhibit reinforcement during the second postnatal week. The appearance of reinforced responses is associated with a change in the early phase of the SCR activity cycle characterized by progressive shortening of the absolute unresponsive period. The distinctive electrographic characteristics of SCR’s in neonatal animals are attributed to summation of 10 to 20msec postsynaptic potentials generated in densely packed apical dendrites of pyramidal neurons and dendrites of embryonic Cajal-Retzius cells. Regression of the dendritic system of the latter cells, decrease in apical dendritic density, increase in conduction velocity of axons in the molecular layer and changes in the SCR-activity cycle are considered the major factors associated with maturation of superficial neocortical neuropil. l This study was supported in part by the National Institute of Neurological Diseases and Blindness (B-1312 CZ), the United Cerebral Palsy Research and Educational Foundation (R-133-60), and the Paul Moore Neurosurgical Research Gift. The senior author is a Sister Elizabeth Kenny Foundation Scholar, Dr. Carmichael is a Visiting Fellow in Neuropharmacology, NIH-USPHS, and Dr. Housepian holds a Postdoctoral Fellowship of the Parkinson’s Disease Foundation. We wish to acknowledge the skillful technical assistance of Mrs. C. De Gorin in preparing the histological material and Mr. P. Huber in preparing the microphotographs. 324

IMMATURE

NEOCORTEX

325

Introduction

This report initiates a series devoted to the developmental physiology of cat cerebral cortex and is primarily concerned with an analysis of the changing structural and physiological properties of neuronal organizations involved in the production of surface-negative responsesevoked by local cortical stimulation (1). Superficial cortical responses(SCR) similar to those described here have been intensively studied in adult animals and man, but as yet there is little agreement concerning their origin and nature or the mechanismsinvolved in their spread along the neocortical surface (27). The SCR has been identified as a conducted action potential in apical dendrites and their divergent branches (7), a graded, decrementally conducted responsein apical dendrites (8) and a propagated responsein tangential fibers (4). The resemblancebetween SCR’s and postsynaptic potentials (PSP) of motoneurons ( 14), as well as their common physiological and pharmacological properties (32) has led to a general hypothesis which views all SCR’s as PSP’s and considers them a prototype of the major form of spontaneousand evoked electrical activity recorded from the surface of the brain (21, 27, 32). Other current theories of cortical electrogenesishave also evolved from studies of the SCR and related types of activity (8, 39). Although it has been shown that SCR’s recorded at different distancesfrom the site of stimulation are similarly affected by synaptic blocking agents (16), some distinctions between near and distant responseshave been reported (3). Neglecting for the moment any further consideration of the various interpretations of the SCR, it is evident from this brief survey that an adequate explanation of the processesinvolved in the production and propagation of these responsesis basic to an understanding of the fundamental nature of brain waves (27). In the present study, an attempt has been made to define the physiological and morphological properties of elements involved in the SCR during the postnatal phase of cortical maturation. The data provide information relating to the comparative histogenesisof neocortex and the potential functional capacity of synaptic organizations in its superficial neuropil prior to the development of organized spontaneouselectrocortical activity. They also serve to focus on some characteristics of the SCR which appear to have been obscured by the overwhelming complexity of the mature brain.

326

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

Methods

Observations on the SCR were made in 42 kittens from litters born in the laboratory, someof which were employed in additional studies on the blood-brain barrier to injected y-aminobutyric acid (GABA) (28), and on the effects of topically applied convulsant o-amino acids (6). The number of animals in different age groups were as follows: O-24 hours, 7; l-3 days, 8; 4-6 days, 4; 7-10 days, 7; 11-15 days, 6; 16-21 days, 5; 3-6 weeks, 5. Attempts were made, whenever possible, to use animals from the samelitter at different ages. A small seriesof late fetal kittens delivered near term by cesarean section were also studied. The results obtained in fetuses surviving this and other surgical procedures were quantitatively similar to those found at birth in spontaneously delivered kittens. All kittens were initially anesthetized with ether to permit introduction of tracheal and external jugular vein cannulas after which the exposed skin margins and scalp were infiltrated with procaine and the ether was discontinued. The animals were then immobilized with succinylcholine chloride and artificially ventilated.2 Following craniectomy, stimulating and recording electrodes, held in 3-coordinate manipulators, were placed in different arrays on the exposed suprasylvian gyrus. Stimulating electrodes consisted of a pair of 100 p Teflon-coated silver wires cemented together. The exposed tips of these wires were lowered until gentle contact with the pial surface was secured without visible signs of overt cortical dimpling. Two types of recording electrodes were used during various phases of this study: a 100 p Teflon-coated silver wire, and chlorided-silver wires with 0.2- and 0.5mm ball-tips. In a few instances in which electrodes with smaller tip-diameter (< 25 cl) were employed, the electrographic characteristics of SCR’s were found to be similar to those recorded with 100 u wire electrodes. Monopolar differential recording was employed throughout, the indifferent lead consisting either of a small screw embeddedin the thin bone overlying the frontal sinus or a clip on the posterior neck muscles. Other details as to stimulating and recording techniques were similar to those describedpreviously (30-34). At the end of the experimental period animals in different age groups were deeply anesthetized with pentobarbital sodium (30 mg/kg) for removal of the entire contralateral cerebral hemisphere. The Golgi-Cox 2 The overt where (29).

effects

of succinyIcholine

in neonatal

cats

have

been

described

else-

IMMATURE

NEOCORTEX

327

method (Romanes modification) was found suitable for demonstrating the salient morphological details of cortical neurons in the newborn cat. Elements of particular importance were photographed from 150 to 200 lo coronal or sagittal serial sections. Results Electrograph& Characteristics of Evoked Superficial Cortical Responses from Suprasylvian Gyrus in the Perinatal Period. When recorded with a large ball-tipped (0.5 mm) electrode placed 1.5 mm from the stimulation

FIG. 1. Characteristics of long-duration superticial cortical responses recorded 1.5 mm (A-C) and 4 mm (D) from stimulating electrodes on suprasylvian gyrus. Stimulus frequency O..S/sec,6-20 superposed responses at different stimulus strengths. Negativity upwards in this and all subsequent figures. A and B, from two late fetal kittens; C, 4-day-old kitten; and D, l&hour-old kitten. Duration of SCR’s independent of stimulant strength; threshold of late components in near (C) or distant (D) responses similar to that for the early. Latency of distant responses is not appreciably altered by five- to tenfold increase in stimulus strength. Note augmentation of early component in near responses and late components of distant responses with strong stimuli. Cals: 1OOcps; 0.1 mv.

site evoked SCR’s exhibited electrographic characteristics which differed in a number of respects from those recorded in adult preparations under identical conditions (30-34). In the latter, progressive increase in stimulus strength results in concomitant increase in amplitude of an initial 10 to 20 msecsurface-negativity and the development of a secondsurfacenegativity of similar duration late on the falling phase of the initial component. With a strong stimulus, the second surface-negativity terminates in a long-duration surface-positivity which may be interrupted by a variable seriesof repetitive responses(1, 3). In late fetal and neonatal preparations, progressive increases in stimulus strength evoked graded long-duration (N-80 msec) surface-negative responseswhich, on occasion, exhibited discontinuities on their falling phases (Fig. 1). The latter became more prominent and consistent by the third to fifth postnatal

328

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

days. The .threshold of late components was usually equal to or lower than that for the early (Fig. 1C). SCR’s recorded 4 to 5 mm from the site of stimulation in neonatal preparations developed after a prolonged latency (cf. below) and exhibited graded characteristics similar to those recorded 1 to 5 mm away from the stimulating site. At distant sites, late components appeared more prominent with weak stimuli (Fig. 1D). Typical examples of SCR’s recorded 1.5 to 2.0 mm from stimulating electrodes in kittens ranging in age from 8 hours to 9 days are shown in Fig. 2A-J arranged in order of increasing age. SCR’s evoked in .5- and

FIG. 2. Typical responses recorded with a OS-mm tip-diameter electrode placed l-2 mm from stimulating wire-electrodes in locally anesthetized, paralyzed kittens, aged: A, 8 hours; B, 12 hours ; C, 18 hours; D, 1.5 days; E, 2 days; F, 2.5 days; G, 4.5 days; H, 7 days; I and J, 9 days; K, 5 weeks; L, 6 weeks. Note long duration of SCR’s and variable relationship of early and late components in A-J. Further explanation in text. Gals: 1COcps.

IMMATURE

NEOCORTEX

329

6-week-old kittens are shown in Fig. 2K, L. All the responseswere recorded from mid-suprasylvian gyrus with the same stimulating-recording electrode combination(0.5mm ball-tipped recording electrode). This illustration stressesthe variability in the overt characteristics of SCR’s, as well as their long duration in neonatal preparations. Clear dissociation between the initial 10 to 15 msec surface-negative component in the SCR and later components is seenas early as the third postnatal week and by the fourth to fifth weeks the adult pattern is fully established (Fig. 2K). To determine whether a clearer dissociation between early and late components in the SCR could be recorded at birth in the cat, a series of experiments were designed in which SCR’s recorded as shown in Fig. 2 were compared with those recorded with a O.l-mm wire electrode virtually in contact with the stimulating electrodes (Fig. 3A). SCR’s recorded at

FIG. 3. Comparison of SCRrecordedat 1.5 mm with a large (0.5 mm) ball-tipped electrode (upper channel) and SCR recorded “at the site of stimulation” with a O.l-mm wire electrode in a S-hour-old kitten. A-D, wire electrode at same position, stimulus strength increased to reveal development of late components a, b. Highfrequency oscillation (a) an inconstant finding in perinatal preparations. Cals: 100 cps; 0.1 mv.

the site of stimulation with wire electrodes showed entirely different characteristics from those observed 1 mm distant. At the site of stimulation a 10 to 15 msec initial surface-negativity was recorded that was sharply demarcated from later components. Other characteristics of SCR’s observed are shown in Fig. 3B-D. Increase in stimulus strength resulted in augmentation of the initial surface-negativity and the development of a second surface-negative component which, on occasion, appeared to be compoundedof a variable seriesof high-frequency oscillations (Fig. 3B, a). This was followed by a third surface-negativity of 15 to 20 msec duration (Fig. 3B, b). When the second component was devoid of the superimposedhigh-frequency oscillations, the SCR recorded at the site of stimulation consistedin a prominent initial 10 to 15 msecsurface-negativity which was interrupted late on its falling phase by a double surface-negativity of low amplitude (Fig. 3D). The double-negativity was often succeeded

330

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

by a more prolonged residual negativity whose characteristics were not defined in these experiments. Failure to detect the complex sequence of distinct 10 to 20 msec surface-negative potentials shown in Fig. 3D with large recording electrodes at distances greater than 1 mm from the site of stimulation is only partly accounted for by differences in the surface areas of the large and small recording electrodes. SCR’s recorded simultaneously with both types of electrodes are shown in Fig. 4. In this experiment a 0.5mm

C

34 57mm

mm FIG.

4

FIG.

5

4. Characteristics of SCR’s at different distances from stimulating electrodes on suprasylvian gyrus in an g-hour-old kitten. Upper channel recordings made with large electrode (0.5 mm) the center of which was placed 1 mm from stimulating electrodes. Lower channel responses recorded with wire electrode at sites indicated at left. Note similar characteristics of responses in C, and fall-off of early component at 4 mm in D. Further explanation in text. Cals: 100 cps; 0.1 mv. FIG. 5. .Relationship between near (upper channel) and distant (lower channel) responses in two late fetal kittens. A, graded responses at near (1.5 mm) and distant (4 mm) sites from stimulating electrodes. Stimulus evoking small near SCR produces detectable distant response after I.5msec latency (propagation velocity, 0.27 m/set). B, characteristics of distant SCR’s to strong stimuli evoking maximal near SCR’s. Note progressively increasing latency of distant responses (mean propagation velocity, 0.25 m/set). Gals: 100 cps; 0.1 mv. FIG.

IMMATURE

NEOCORTEX

331

tip-diameter recording eIectrode was “fixed” 1.0 mm away from the site of stimulation and a O.l-mm wire electrode was moved outwards from the stimulating wires in 1.0 mm steps. At the site of stimulation a distinct 15msec response was evoked with weak surface stimulation, whereas at 1.0 mm a long-duration SCR was recorded with the large electrode (Fig. 4A). When both electrodes were equidistant from the site of stimulation, the initial component of SCR’s recorded with the wire electrode was of greater duration than that evoked at 0.1 mm, but was still demarcated from late components (Fig. 4B). SCR’s recorded with the wire electrode at 2 mm were entirely similar to those recorded at 1 mm with the large electrode (Fig. 4C). At distances greater than 2 mm from the site of stimulation, SCR’s recorded with a O.l-mm wire electrode (Fig. 4D) were of long duration and exhibited no discontinuities. Suffice it to say that smooth-contoured, long-duration SCR’s were recorded beyond 2 mm in neonatal animals with electrodes of tip-diameter less than 25 u. Summarizing the results of these studies, it is apparent that a prominent initial surface-negativity of IO to 20msec duration can be recorded in neonatal cat cortex as in mature cortex. Whereas in the latter a sharp demarcation between the initial and late components in the SCR can be recorded at distances greater than 5 mm from the site of stimulation (Fig. 7E), in neonatal cortex clear differentiation between early and late components is detected only at distances less than 1 mm from the stimulating electrodes. Beyond this, SCR’s of long duration are recorded due to the rapid build-up of late components. It is concluded from this analysis that long-duration SCR’s evoked in immature cortex (at distances comparable to those at which SCR’s of lo- to 20-msec duration are recorded in mature cortex) result from summation of a series of temporally dispersed unit lo- to 20-msec surface-negative potentials. Propagation of SCR Along Surface of Suprasylvian Gyms at 0 to 5 Days. Graded responses recorded at sites remote from stimulating wires developed with a latency determined by the distance between recordingstimulating electrodes (Fig. 1D). The relationship between near and distant3 responses to increasing strengths of stimuli and recorded in a late fetal kitten is shown in Fig. 5A. With constant relatively strong 3 The terms, near and distant, as employed here refer to the distance between stimulating and recording electrodes and no other significance is implied, Near responses are arbitrarily defined as those recorded within 2 or 3 mm of the site of stimulation, and distant responses, those recorded at distances greater than 3 mm

(16).

332

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

stimuli, sufficient to evoke large amplitude near SCR’s, the responses at distant sites developed with progressively increasing latency and with disproportionate loss of early components (Figs. SA, 6). In kittens 0 to 5 days old, SCR’s were detectable up to 5 to 7 mm from the site of stimulation. In none of the kittens studied in this age group were distant responses evoked that were of greater magnitude than those recorded within 2 mm of the stimulating electrodes. Propaga,tion at 7 to 12 Days. Distant SCR’s evoked in kittens 7 to 14 days old exhibited dramatically different characteristics from those observed in animals 0 to 5 days old. Linear increases in latency with

FIG. 6. Spread of SCR in a 5-hour-old kitten. Upper channel, near SCR’s recorded 1.5 mm from stimulating electrode ; lower channel, distant response at 4 mm, A; 5 mm, 33; and 6 mm, C (five superposed records; stimulus frequency 0.5/set). In D, sample records from A-C superposed to show latency changes of distant responses. Minimal response at 6 mm of doubtful significance (propagation velocity approximately 0.4 m/set) .

increasing distance from stimulating electrodes were still apparent, but distant SCR’s were often of greater magnitude than those recorded 1 to 2 mm from the site of stimulation (Fig. 7). This phenomenon described as reinforcement by Brooks and Enger (3) has been studied by the latter in considerable detail in adult animals. It was detectable as early as the seventh postnatal day, but found to be more consistently demonstrable in animals 8 to 12 days old. Reinforcement was not observed in poor preparations requiring extraordinarily high stimulus intensities for near SCR’s and exhibiting rapid fall-off of distant responseswith small displacements of the recording electrode. Of the various patterns observed, the most typical are shown in Fig. 7. In kittens 8 to 12 days old SCR’s increased in amplitude at 3 to 5 mm and progressively decreased beyond this. In animals 14 to 21 days old a second locus of reinforcement was often observed at 8 to 9 mm, but was less constant than that at 3 to 5 mm. The electrographic characteristics of reinforced distant responseswere similar to distant SCR’s recorded in animals 0 to 5 days old, i.e., long latency to onset and slow build-up to

IMMATURE

333

NEOCORTEX

peak amplitude, as compared with near responses. Propagation velocities of SCR’s in kittens ranging from 7 to 14 days of age were only slightly greater (0.4-0.5 m/set) than those calculated in perinatal preparations. Velocities of 0.6 to 0.7 m per second were found in 3-week-old kittens, the upper value being similar to that reported in adult cats (3, 4, 7). Utilizing the commonly accepted method of calculating latency from shock artifact to onset of response, no evidence was obtained that distant AteD)

B(lOD)

D (1401

C(l2Dl

E 140

ADULT

FIG. 7. Changing characteristics of distant SCR’s during second postnatal week. A-D, relationship between near (upper channel) and distant (lower channel) responses recorded at distances from stimulating electrodes indicated at left in each series; ages shown above. A, SCR’s at 5 and 3 mm larger than at 2.5 and 4 mm. magnitude of response B, reinforced responses at 4 and 6 mm. C, note extraordinary at 4 and 6 mm. D, reinforced SCR at 8 mm ; latency about 25 msec. In C, records at bottom of column taken before and after topical application of 1 “/o GABA to near electrode site. In this record, upper channel shows abolition of surface-negativity and unmasking of surface-positivity after GABA; lower channel shows two E, comparison of responses at indicated perfectly superposable distant responses. distances in a 14-day-old kitten and adult cat studied under identical conditions. Wire electrode (0.1 mm) employed to record response at 0.3 mm in kitten. Compare characteristics of responses at 1 mm. Duration of the distant response in adult preparation is similar to that of near response. Gals: 100 cps; 0.1 mv in A; 0.3 mv in B-E; that for D shown in E.

334

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

reinforced SCR’s propagated at velocities greater than nonreinforced responses recorded close to stimulating electrodes.4 The lack of direct relationship between near and distant SCR’s was established by the finding that abolition of SCR’s by topical application of GABA to near electrode sites did not alter the characteristics of distant responses (Fig. 7C, bottom record). Similar findings have been reported in adult cats (23). Activity Cycle Characteristics of SCR in Immature Cortex. The remarkable stability of SCR’s evoked in neonatal cat cortex permitted a

FIG. 8. A, early phase of SCR activity cycle in a S-hour-old kitten. Recording electrode 1.5 mm from stimulating electrodes. Two series are shown at different strengths of stimulation. The conditioning (C) and testing (T) responses in each series were of equal magnitude. The broken lines a’ and a are drawn through the peaks of responses evoked by weak and strong pairs of stimuli, respectively. The first records in each series show C and T responses in isolation, superposed. Thereafter, C-T intervals are as indicated. No increment in conditioning SCR is detectable at 5-msec C-T stimulus intervals in either series. At slightly greater intervals a small increment in conditioning SCR is observed. B, early phase of SCR activity cycle in a jr-day-old kitten. Recording electrode 2.0mm from stimulating electrodes. First records in series show equal C and T responses in isolation superposed. Broken line drawn through peak of these responses. When C-T interval is 1 msec, a significant increment in conditioning SCR is detectable. With a 4-5 msec C-T interval, recovery is greater than that obtaining at 8 msec in 5-hour-old kitten (A). Cals: 1OOcps; 0.1 mv in A; 1.5 mv in B.

relatively precise analysis of their activity cycles as demonstrated by paired conditioning C-testing T shocks to the cortical surface (8, 34). Figure 8 shows two series of experiments with stimuli of different strength in which the conditioning and testing stimuli in each series were adjusted to give SCR’s of equal amplitude. The peak amplitude of C and T responses in isolation and superposed is indicated by the broken lines a and a’ for responses to strong and weak stimuli, respectively. No significant 4 Differences in propagation which peak-response latencies

velocity have been reported in adult were utilized in the calculations (3).

preparations

in

IMMATURE

NEOCORTEX

335

increment in the conditioning SCR was detectable in late fetal or neonatal animals until C-T stimulus intervals were greater than 4 or 5 msec (Fig. SA). At slightly greater intervals (7-8 msec), the testing stimulus evoked a small response which added to the conditioning SCR. Identical activity cycles were recorded at different stimulus strengths (Fig. 8A, a

FIG. 9. Early phases of near and distant SCR’s in a 12-hour-old kitten. A and B responses recorded 1.5 mm from stimulating electrodes. In A, C > T. Both responses shown in isolation and superposed. Broken line drawn through peak of C response. Increment in C response detectable with 5-msec C-T stimulus interval. In B, T > C; both responses shown in isolation and superposed. Broken line drawn through peak of T response. Reduction in T response with S-msec C-T interval is attributable to residual depression of elements activated by C stimulus. Considerable recovery seen at 6-msec C-T intervals. C, SCR’s recorded 5 mm from stimulating electrode. Conditioning SCR shown in isolation as first response of series. With C = T, first significant increment is detectable at 4.5msec stimulus intervals; example of further temporal summation with 11-msec C-T interval. Cals: 0.1 mv; 100 cpsin A and B ; ll3lOcps in c.

and a’). The prolonged absolute unresponsive period (AUP) in neonatal kittens was also demonstrable when C and T were adjusted so that C > T (Fig. 9A) or T > C (Fig. 9B). With C > T, no change in the conditioning SCR was observed at C-T intervals less than 5 msec. The SCR evoked by the strong stimulus delivered 5 msecafter the weaker C stimulus (Fig. 9B) was less than the testing responseunconditioned, indicating that only a small fraction of the elementscontributing to the conditioning

336

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

response were capable of adding to the activity of unconditioned elements involved in the large testing response. Of particular importance was the finding that the AUP was identical in any one particular preparation for both near and distant responses (Fig. 9C). The AUP in perinatal cats was followed by a phase of depressed responsiveness of variable duration (20-200 msec). Late components of near SCR’s appeared to suffer greater depression than the early, whereas differential depression was not readily detectable in distant responses. In contrast to the relatively long AUP found in perinatal preparations, that in kittens 7 to 14 days old was often less than 1 msec and rarely greater than 1.5 msec (Fig. 8B). This change in responsiveness became evident

FIG. 10. Comparison of early phase of activity cycles of near (1.5 mm) and distant (5.0mm) SCR’s in a lo-day-old kitten. Conditioning response (C) larger than testing response (T). Numbers above records indicate stimulus interval. Minimal increment in both near and distant responses at 1.5-msec stimulus intervals, marked increment at 3 to IO-msec intervals. Note extraordinary magnitude of smoothly summated distant responses at IO-msec C-T stimulus interval. Delayed recovery still evident at 16 msec. Gals: 100 cps; 0.3 mv.

as early as the fifth postnatal day. The early phase of SCR-activity cycles recorded at near and distant sites in a lo-day-old kitten is shown in Fig. 10. In this experiment conditioning responses were made larger than the testing to insure that all elements involved in the production of the latter would be activated by the conditioning stimulus. A distinct increment in near and distant SCR’s is detectable at 1.5msec intervals and progressive increments at 3- and IO-msec intervals, respectively. Summation of distant SCR’s producing responses of extraordinary duration and amplitude were commonly observed in animals 7 to 21 days old.

IMMATURE

337

NEOCORTEX

Structural Characteristics of Neocortical Neurons in Newborn Cat.5 The relevant features of neocortical neuronal architecture in a cat 5 hours old are shown in Fig. 11. The electrographic characteristics of near and distant SCR’s from this preparation have been noted above (Fig. 6). Small, medium, and large pyramidal neurons possess well-developed apical dendrites which emerge from cell bodies whose diameters range from 10 to 25 ~1. The proximal unbranched portions of the apical dendrites are of considerable thickness relative to cell body diameter. Branching of middle or distal segments is minimal, but when this occurs radial orientation of branches is maintained. Tangential branches of apical dendrites in their proximal or middle segments have never been observed at birth in the cat, although a few delicate processes less than 50 p were occasionally discerned in small superficially located pyramidal neurons. Apical dendrites extend into the molecular layer where large numbers of them, densely packed, terminate within 25 p of the pial surface. Others appear to end in contact with the latter. Tangential spread in the molecular layer of apical dendrites of medium and giant pyramidal cells is rarely greater than 100 p, but some small, superficially located neurons give rise to apical dendrites whose tangential branching in the molecular layer may occasionally approach 150 to 200 ~1 (38). The distal segments of apical dendrites exhibit minute, regularly spaced bulbous dilatations, but these are rarely observed on middle and proximal segments. Thornlike projections (spines or gemmules) on apical dendrites are not demonstrable in the newborn cat. The most conspicuous feature of the pyramidal cell dendritic system in the neonatal period is the relatively poor development of basilar dendrites of medium and large pyramids. In contrast, some small pyramidal neurons in the submolecular layer may have basilar dendrites three to four times the diameter of their cell bodies. Basilar dendrites of the larger pyramids are wisps of twiglike ramifications rarely longer than 25 ~1. Basilar dendrites of superficially located small (star?) cells are absent, but the terminal branch “bushels” of these elements are well developed and ramify in the molecular layer. Descending axons of pyramidal neurons of all sizes are well developed but are relatively devoid of collaterals. Some ascending axons of unknown origin often appear to be intimately related with apical dendrites of pyramidal neurons throughout their entire course. 5 We wish to thank Dr. Rafael Lorente de N6 for reviewing presented here and for his invaluable assistance in facilitating

the histological their interpretation.

data

FIG. 11. Golgi-Cox preparations of neurons in suprasylvian (A and B) and anterior sigmoid gyrus (C and D) of S-hour-old kitten. SCR’s recorded in this animal shown in Fig. 6. A, cluster of medium and large pyramidal neurons. Apical dendrites extend to within 25-501 of pfal surface. Note absence of lateral branches. Axons of two neurons clearly shown extending downward. Basilar dendrites at this stage are short, thin processes. B and C, embryonic Cajal-Ret&s neurons in molecular layer. D, giant pyramids in motor cortex with massive apical dendrites but poorly developed basilar dendritic system. Further description in text. Magnification in all photomicrographs, 200x.

IMMATURE

NEOCORTEX

339

The general characteristics of Cajal-Retzius cells in the molecular layer of the neonatal cat cortex are shown in Fig. llB, C. The structural details of these elements are entirely similar to those observed in the newborn human infant (5, 9). Dendrites of these cells generally course tangentially for varying distances (So-200 u) in the middle and lower third of the molecular layer. Emerging at right angles to these tangential elements are numerous radially oriented branches which terminate near to or at the pial surface. Such right-angle branches may be spaced as closely as 5 to 10 u apart and arise from small varicosities on the tangential elements. Distinctions between axons and dendrites of these cells are often difficult to establish. Some of their identifiable axons intertwine with densely packed apical dendrites of pyramidal neurons and may be followed for 100 to 200 u before they are lost in a maze of apical dendrites. The growth of basilar dendrites of medium and large pyramidal neurons appears to gain momentum within a few days after birth and by the third postnatal day numerous short elements (X-75 u) of small caliber relative to cell body diameter begin to arborize extensively (Fig. 12A). An accelerated phase of basilar dendritic growth occurs during the early part of the second postnatal week (Fig. 12B) and continues to the end of the third postnatal week. At the end of this period basilar dendrites of extraordinary length and diameter are readily seen in all neocortical areas (Fig. 12C). During this period “spines” become prominent on apical and basilar dendrites and axonal proliferation is extensive. Cajal-Retzius cells in the molecular layer undergo “regressive” changes by fifth to eighth postnatal days, which eventually result in the loss of the numerous radially oriented dendritic ramifications characteristic of the perinatal period (5, 10, 11). Apart from changes in volume, apical dendrites of pyramidal neurons in kittens 7 to 21 days old have characteristics similar to those at birth. The general characteristics of neocortical neurons described above pertain largely to those in suprasylvian and pericruciate cortex. Other details as to differences in neurons in different neocortical regions and phylogenetically different types of cortex will be described elsewhere along with the quantitative changes in neuronal morphology accompanying cortical maturation in the cat. Discussion

The superficial cortical response was originally described by Adrian (1) who assigned it to the activity of “nerve cells with dendrites running

340

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

laterally, e.g., horizontal cells of Cajal or the superficial branches of apical dendrites of pyramidal cells.” The extent to which the present study contributes to a further understanding of the SCR will now be briefly considered with respect to the extant literature on this subject.

FIG. 12. A, examples of small and medium pyramidal neurons from suprasylvian gyrus of S-day-old kitten. Basilar dendrites beginning rapid phase of growth. B, further elaboration of basilar dendritic system in &day-old kitten. C, large pyramidal neurons from anterior suprasylvian gyrus of 21-day-old kitten. Note extensively developed basilar dendrites and spines on apical dendrites. Further description in text. Magnification 250X.

IMMATURE

NEOCORTEX

341

SCR’s evoked at various distances from the site of stimulation in immature cortex have fundamentally similar characteristics, yet abolition of SCR’s recorded near stimulating electrodes by topical GABA does not alter responses recorded a few millimeters away. These results confirm previous findings that the action of GABA is confined to superficial elements involved in the generation of the SCR (30-33), but not in its spread to distant sites (23). The dual nature of the processes of generation and spread is further supported by the demonstration of variations in SCR amplitudes at different loci. These observations preclude the possibility that the SCR is both generated in and conducted along a single species of tangential fibers (4). A mechanism involving conduction along tangential branches of apical dendrites or their divergent branches (7) is rendered untenable by the limited tangential branching (SO-100 p) of these elements in the molecular layer and the absence of lateral apical dendritic branches in submolecular regions of cortex in the newborn cat. Suffice it to say that the available data are accounted for in terms of an hypothesis proposed earlier (32) that the SCR is postsynaptically generated in apical dendrites by conductile pathways of different lengths. Although the anatomical data do not permit positive identification of axodendritic synapses, it is curious that small protuberances are observed largely on the distal segments of apical dendrites in the newborn cat as they are in the newborn human infant (5, 9). Whether these structures are artifacts or poorly developed “spines” which have been recently identified as synaptic loci (18) cannot be decided at this time. However, the failure to observe axodendritic synapses in Golgi-Cox preparations is of doubtful significance since synapses on cortical neurons have been clearly demonstrated only with special histochemical methods (2) or the electron microscope (18, 26). The existence of axodendritic synapses in immature cortex is compelled by the physiological data presented here. The interpretation of other data on the SCR need not be compromised by the lack of morphological data on their ultrastructure in immature cortex. The most noteworthy characteristic of SCR’s evoked in immature cortex is their relatively long duration when recorded at distances (> 1 mm) comparable to those at which 15 to 20-msec responses are ordinarily observed in mature cortex. Similar long-duration responses have been reported in the newborn rabbit ( 15). Since 15 to 20 msec responses are clearly detectable at the site of stimulation in perinatal cats, differences in over-all duration of distant responses cannot be attributed

342

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

to differences in the kinetics of the ionic processes responsible for the SCR. An adequate explanation may be formulated on the basis of the relative density of dendritic elements in immature and mature cortex. A decreased density in mature cortex results from the over-all change in cortical volume secondary to the growth of nonneural elements, proliferation of basilar dendrites, axon collaterals, and increases in cell body diameter. Furthermore, the dendritic system of embryonic Cajal-Retzius cells undergoes regressive changes during cortical maturation. Thus, although there may be fewer synapses on dendrites in immature than mature cortex, this is likely to be balanced by differences in dendritic density. At extremely short distances (0.142 mm) temporal dispersion in horizontal axons making contact with apical dendrites of pyramidal neurons and dendrites of Cajal-Retzius cells can be expected to be negligible and Despite this synchronization synaptic activation relatively synchronous. which results in a distinct 15 to 20-msec surface-negativity (PSP), late components of low amplitude may still be detectable (Fig. 3). Postsynaptic activity in embryonic Cajal-Retzius cells could account for some of the characteristics of late components, especially the high-frequency oscillations that are occasionally superposed on these responses in the perinatal period. The relative magnitude of late components increases with increasing distance from the site of stimulation until they come to surpass the initial component. Progressive fall-off of early components in SCR’s with distance may be attributable to a reduction in the number of long axons traversing distances of 5 to 8 mm. In view of the linear changes in latency of the earliest response detectable at these distances, conduction in a relatively homogenous group of axons seems a reasonable assumption. The spread of late components may involve activity in a second more heterogenous group of fibers with a more circuitous trajectory (25) or the conductile pathway to distant dendritic sites may be interrupted by one or more synapses (3). A possible clue as to the nature of the processes involved in the production of distant responses is provided by the finding that the latter exhibit reinforcement sometime during the second postnatal week, a period characterized by a rapid expansion of the basilar dendritic and axon collateral system of neurons. If descending axon collaterals from tangential fibers establish effective contact with basilar dendrites and cell bodies of neurons in submolecular regions during this period, relayed activity in recurrent axons from these elements may generate PSP’s in apical dendrites which summate with earlier ones evoked at different sites in the same population of dendrites by the more

IMMATURE

NEOCORTEX

343

direct tangential pathway. Although further speculation on the mechanisms responsible for reinforced distant responses must await additional data, it should be noted that no evidence has been obtained in this study on immature cortex to indicate that distant reinforced responses are initiated by more rapidly conducting pathways than those which generate SCR’s close to the site of stimulation as has been inferred by Brooks and Enger (3) from studies on adult cats. Whether or not SCR’s in immature cortex exhibit reinforcement, the first potential change signaling postsynaptic activity at all sites where the onset can be clearly detected is initiated by a pathway(s) conducting impulses at 0.2 to 0.4 m per second in perinatal animals and 0.5 to 0.6 m per second in kittens 2 to 3 weeks old. The prolonged absolute unresponsive period in the activity cycle of SCR’s evoked in perinatal cortex is of considerable interest both with respect to the interpretation of these responses advanced here and elsewhere (2 1, 27, 30-34) and the changes in the properties of superficial cortical elements during the first postnatal week. Since PSP’s are evoked in membrane which lacks regenerative action and absolute refractoriness (20)) it is unlikely that the AUP results from an inability of transmitter substance(s) to induce an electrogenic effect in apical dendrites 4 to 7 msec after the onset of conditioning postsynaptic activity. Delayed synthesis of transmitter(s) coupled with refractoriness in the presynaptic conductile pathway would appear to be a more satisfactory explanation. The extent to which absolute refractoriness in the latter contributes to the AUP is not known. It is of interest that the AUP shortens to less than 1.5 msec by the end of the first postnatal week, at a time when no significant change is detectable in the conductile properties of these elements. Assuming, therefore, a minimal AUP of 1 to 1.5 msec in the conductile elements at birth, the additional 3 to 5 msec could represent the time required for activation of new transmitter substance(s). Implicit in this assumption is the notion that in the immediate neonatal period the enzymatic mechanisms subserving the manufacture, storage, and release of transmitters are likely to be operating at a relatively low level of activity. Although the nature of these biochemical processes in cerebral cortex are unclarified, some support for this may be found in the literature on the biochemistry of the developing nervous system (17, 35). Acceleration of biochemical processes involved in synaptic transmission could be expected to increase markedly the effectiveness of the second of two closely timed volleys. The over-all change in excitability, as

344

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

reflected in AUP changes occurs pari passu with the appearance of distant reinforced SCR’s, i.e., after the first postnatal week. This suggests that temporal summation, as well as spatial summation of PSP’s generated in apical dendrites may contribute to the development of distant responses of great magnitude. The morphological features of neocortical neurons in the newborn cat indicate that they have reached a relatively advanced stage of development in this species at birth, as in other immature-born mammals (5, 24) including the newborn human infant (5, 9). The difference in the development of apical and basilar dendritic systems in newborn cat is especially noteworthy and appears to be somewhat more pronounced than in the mouse and rat (24) and newborn human infant (9).6 These histological findings taken together with the physiological data on the SCR suggest that at least in the immediate neonatal period postsynaptic activity in the vast majority of cortical neurons is generated predominantly in their apical dendrites since these elements provide the major part of the available surface area for synaptic contacts. Rapid expansion of the basilar dendritic system of pyramidal neurons in the cat proceeds during the second postnatal week and this appears to be associated with a variety of changes in spontaneous and evoked cortical potentials in this species ( 19, 37). It can be expected that the marked increase in the available synaptic receptor surface on neurons which results from this developmental change will exert profound effects on their discharge characteristics and on the magnitude of different components of the surface potentials which comprise the summated activities of large populations of cortical neurons (27). It is of interest to note that evoked primary cortical responses to somatic sensory (37) and optic nerve stimulation (22) in immature-born animals at birth differ significantly o Although we will take up the problem of the histogenesis of neocortex in the cat in greater detail in another communication, it is pertinent to note that in recent studies some attempts have been made at a quantitative analysis of the developmentaf changes in cortical neuronal relations in the newborn rat (13) and rabbit (36). Apart from differences in the temporal pattern of postnatal histogenesis, the over-all changes in neuronal morphology are similar to those described here for the cat. Surprising findings in these studies are the failure to demonstrate cortical neurons with the Golgi-Cox method in the newborn rat, the modest development of apical dendrites in 6- to It-day-old rats and the complete absence of dendrites in newborn rabbit. These data are contrary to the findings of other workers (5, 24, and Lorente de No, personal communication) who have observed well-developed apical dendrites of cortical neurons at birth in these species.

IMMATURE

345

NEOCORTEX

from those recorded in the adult animal with respect to the polarity of the initial component in surface recordings. The prominent initial surfacenegativity of these responsesmight thus be a reflection of the underlying difference in the nature of the activity evoked in cortical neurons by primary thalamocortical projection pathways in the neonatal period. A reasonable interpretation is that the synaptic activity generated in the cortical depths at this stage is relatively weak and likely to be swamped by summatedaxodendritic PSP’s developing in superficial cortical regions. In view of the remarkable development of axodendritic synaptic organizations in the superficial region of neocortex in the immature cat, the question arises whether these organizations may participate in the elaboration of behavioral activities characteristic of the immediate neonatal period (12).’ Although an adequate answer to this requires more information than is currently available, the data presented here suggest that the functional capacity of immature neocortex may be far greater than has been inferred from studies of its spontaneouselectrical activity. Addendum

Since completion of the present studies, Schade and Baxter have published an analysis of the changes during growth in the volume and surface area of cortical neurons in the rabbit (Exptl. Neurol. 2: 158-178, 1960). It was stated that “dendrites are hardly countable” in the newborn rabbit, but “even at birth a number of neuronsshow apical dendrites; they can be observed in Golgi-Cox preparations as thin threads.” GolgiCox material on the newborn rabbit neocortex (to be presented elsewhere) reveals apical dendrites of pyramidal neurons that are comparable in volume and number to those described here for the newborn cat (Fig. 11). References 1.

ADRIAN,

E. D.,

127-161, 2.

3.

ARMSTRONG, 137: 10-11,

The

spread

of activity

in the

cerebral

cortex.

J. A., and J. 2. YOUNG,

Physiol. 88:

End-feet

in the cerebral

cortex.

J.

Physiol.

1957.

V. B., and P. S. ENGER, Spread of directly evoked cat’s cerebral cortex. J. Gen. Physiol. 42: 761-777, 1959.

BROOKS,

/.

1936.

responses

in

the

r This problem and others relating to the ontogenesis of complex behavioral patterns in animals and man are discussed in considerable detail in the “Third Conference on Central Nervous System and Behavior,” M. A. B. Brazier (ed.), Josiah Macy, Jr. Foundation, New York, 1960 (in press).

346 4. 5.

6.

PURPURA,

CARMICHAEL,

AND

HOUSEPIAN

B. D., “The Mammalian Cerebral Cortex,” London, Edward Arnold, Ltd., 1958. CAJAL, S. RAMON Y, “Histologie du systeme nerveaux de l’homme et des vertebres,” Paris, A. Maloine, 1909. CARMICHAEL, M. W., E. M. HOUSEPIAN, and D. P. PURPURA, Effects of ‘convulsant’ co-amino acids during cortical maturation. Federation Proc. 19: 267,

BURNS,

1960. CHANG,

H.-T., Dendritic potential of cortical neurones as produced by direct electrical stimulation of the cerebral cortex. J. Neurophysiol. 14: l-21, 1951. 8. CLARE, M. H., and G. H. BISHOP, Properties of dendrites; apical dendrites of the cat cortex. Electroencephulog. and Clin. Neurophysiol. 7: 85-98, 1955. 9. CONEL, J. L., “The Postnatal Development of the Human Cerebral Cortex. I. Cortex of the Newborn,” Cambridge, Mass., Harvard University Press, 1939. 10. CONEL, J. L., “The Postnatal Development of the Human Cerebral Cortex. II. Cortex of the One-Month Infant,” Cambridge, Mass., Harvard University Press, 1941. CONEL, J. L., “The Postnatal Development of the Human Cerebral Cortex. IV. 11. Cortex of the Six-Month Infant,” Cambridge, Mass., Harvard University Press, 1951. 12. DASHKOVSKAYA, V. S., [The first conditioned reactions in newborn infants under normal and in certain pathological conditions.] (Russian text). Zhur. 7.

Vysshei 13. 14. 15.

16.

17.

18. 19.

Nervnoi

21. 22.

im Pavlov

3: 247-259,

1953.

Electroencephulog. and Clin. Neurophysiol. 3: 449-464, 1951. FAN, S.-F., A study of the electrical activity of cerebral cortex of rabbit during the period of postnatal development. Acta Physiol. Sit&u (Shanghai) 21: 51-62, 1957. FAN, S.-F., and T. P. FENG, Concerning conduction and electrical excitability in the terminal portion of the apical dendrites of the pyramidal neurons. Acta Physiol. Siti (Shanghai) 21: 423-434, 1957. FLEXNER, L. B., Enzymatic and functional patterns of the developing mam-

malian brain. In “Biochemistry of the Developing Nervous System,” H. Waelsch, (Ed.), New York, Academic Press, 1955. GRAY, E. G., Axo-somatic and axo-dendritic synapses of the cerebral cortex: An electron microscope study. J. Amt. 93: 420-433, 1959. GROSSMAN, C. G., Electra-ontogenesis of cerebral cortex. A.M.A. Avch. Neurol. Psych&.

20.

Deyatel’nosti

EAYRS, J. T., and B. GOODLEAD, Postnatal development of the cerebral cortex in the rat. J. Amt., Loud. 93: 385-402, 1959. ECCLES, J. C., Interpretation of action potentials evoked in the cerebral cortex.

74: 186-202,

1955.

GRUNDFEST,H., Electrical inexcitability of synapses and some consequences in the central nervous system. Physiol. Rev. 37: 337-361, 1957. GRUNDFEST, H., Electrophysiology and pharmacology of dendrites. Electroencephalog. and Clin. Neurophysiol. Suppl. 10: 22-41, 1958. HUNT, W. E., and S. GOLDR~NG,Maturation of evoked response of the visual cortex in postnatal rabbit. Electroencephulog. and Clin. Neurophysiol. 9: 465471, 1951.

IMMATURE

NEOCORTEX

347

23. JASPER, H. H., S. GONZALU, and K. A. C. ELLIOT, Action of y-aminobutyric acid (GABA) and strychnine upon evoked electrical responses of cerebral cortex. Federation Proc. 17: 79, 1958. 24. LORENTE DE No, R., Studies on the structure of the cerebral cortex. 1. Psychol. u. Neural. 45: 381-438, 1933. 25. OCHS, S., The direct cortical response. J. Neurophysiol. 19: 513-523, 1956. 26. PALAY, S. L., Synapses in the central nervous system. J. Biophys. Biochem. Cytol., Suppl. 2: 193-206, 1956. 27. PURPURA, D. P., Nature of electrocortical potentials and synaptic organizations in cerebral and cerebellar cortex. Inter. Rev. Neurobiol. 1: 47-163, 1959. 28. PURPIJRA, D. P., and M. W. CARMICHAEL, Characteristics of blood-brain barrier to systemic y-aminobutyric acid in newborn cat. Science 131: 410-412, 1960. 29. PURPURA, D. P., M. W. CARMICHAEL, and E. M. HOUSE~IAN, Succinylcholineinduced contractures in skeletal muscles of newborn cat. Proc. Sot. Exptl. Biol. Med. 103: 336-338, 1960. 30. PURPURA, D. P., M. GIUDO, and H. GRUNDFEST,Selective blockade of excitatory synapses in the cat brain by y-aminobutyric acid (GABA). Science 125: 1200-1202, 1957. 31. PURPURA, D. P., M. Gram, and H. GRUNDFEST,Components of evoked potentials in cerebral cortex. Electroencephulog. and C&n. Neurophysiol. 12: 95110, 1960. 32. PURPURA, D. P., and H. GRUNDFEST,Nature of dendritic potentials and synaptic mechanisms in cerebral cortex of cat. J. Neurophysiol. 19: 573-595, 1956. 33. PURPURA, D. P., M. GIRADO, T. G. SMITH, D. A. CALLAN, and H. GRUNDFEST, Structure-activity determinants of pharmacological effects of amino acids and related compounds on central synapses. J. Neurochem. 3: 238-268, 1959. 34. PURPURA, D. P., E. M. HOUSEPIAN, and H. GRUNDFEST,Factors affecting activity cycles of apical dendrites. Federation Proc. 16: 102, 1957. 35. ROBERTS, E., “Third Conference on Central Nervous System and Behavior,” M. A. B. Brazier (ed.), New York, Josiah Macy, Jr. Foundation, 1960 (in press). 36. SHADE, J. P., and C. F. BAXTER, Maturational changes in the cerebral cortex. I. Volume and surface determination of nerve cell components. In “Inhibition in the Nervous System and y-Aminobutyric Acid (GABA),” London, Pergamon, 1960 (in press). 37. SCHERRER,J., et D. OECONOMOS,Responses tvoquees corticales somestheseques des mammiferes adulte et nouveau-m? In “Les Grandes Activites du Lobe Temporal,” Masson, Paris, 1955. 38. SHOLL, D. A., “The Organization of the Cerebral Cortex,” London, Methuen; New York, Wiley, 1956. 39. TASAKI, I., E. H. POLLEY, and F. ORREGO,Action potentials from individual elements in cat geniculate and striate cortex. I. Neurophysiol. 17: 454-474, 1954.